Gastrointestinal (GI) helminths pose a significant threat to the livestock industry and are a recognized cause of global morbidity in humans. Control relies principally on chemotherapy but in the case of nematodes is rapidly losing efficacy through widespread development and spread of resistance to conventional anthelmintics and hence the urgent need for novel classes of anthelmintics. Cysteine proteinases (CPs) from papaya latex have been shown to be effective against three murine nematodes Heligmosomoides bakeri, Protospirura muricola and Trichuris muris in vitro and in vivo and against the economically important nematode parasite of sheep Haemonchus contortus. Preliminary evidence suggests an even broader spectrum of activity with efficacy against the canine hookworm Ancylostoma ceylanicum, juvenile stages of parasitic plant nematodes of the genera Meloidogyne and Globodera and a murine cestode Hymenolepis microstoma in vitro. This project focused on tapeworms. Using 2 different rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma and 1 equine cestode Anoplocephala perfoliata I have been able to show that CPs do indeed affect cestodes whether young newly hatched scoleces in vitro (by causing a significant reduction in motility leading to death of the worms) or mature adult worms in vitro (by causing a significant reduction in motility leading to death of the worms) and in vivo (resulting in a significant, but relatively small, reduction in worm burden and biomass), despite no effects on worm fecundity. Although only minimally efficacious against Hymenolepis microstoma and moderately efficacious against Hymenolepis diminuta in vivo, efficacy was enhanced by the synergistic effects of the immune system demonstrated against Hymenolepis diminuta in the non-permissive host. The results offer the possibility that with further refinement, CPs may be developed into broad spectrum anthelmintics that in addition to their marked effects on nematodes also remove any concurrently residing tapeworms.

Reactive oxygen species (ROS) are linked to a range of degenerative conditions in humans, and may cause damage to an array of cellular components. However, it is unclear which cellular target(s) of ROS may primarily account for toxicity during oxidative stress. The sensitivity of iron-sulphur (Fe-S) clusters to ROS makes these candidate determinants of ROS mediated cell killing. Ribonuclease L inhibitor (Rli1p) is a highly conserved protein that is essential in all tested eukaryotes and archaea, but requires Fe-S clusters for its crucial functions in protein synthesis. Herein, the novel hypothesis that ROS toxicity is caused by loss of Rli1p function was tested. Rli1p activity (in nuclear export of ribosomal subunits) was impaired during mild oxidative stress in yeast. In addition, resistance to pro-oxidants was decreased by RLI1 repression and increased by RLI1 overexpression. This Rli1p-dependency was abolished during anaerobicity and accentuated in cells expressing the Fe-S cluster defective Rli1p construct, rli1C58A. The effects appeared specific to Rli1p as overexpression of other essential Fe-S proteins did not increase stress resistance. Methionine sulphoxide reductases (MSRs) and the Mn-superoxide dismutase (Sod2p) are known to help preserve the integrity of Fe-S clusters in cells. Here, these proteins’ antioxidant actions were shown to be at least partly mediated through Rli1p. Resistance to both chronic and acute oxidative stress was Rli1p-dependent. Further experiments indicated that Rli1p-dependent protein synthesis could be a critical target of ROS and, specifically, that Rli1p function may help to protect against ROS-induced mRNA mistranslation. The study indicated that Rli1p function is a primary biological target of ROS action, owing to its essential nature but dependency on ROS-labile Fe-S clusters. Such insights could offer new approaches for combating oxidative stress-related disease.

The DEFA1A3 locus on human chromosome 8p23.1 exhibits extensive copy number variation; individuals have between 3-16 copies of DEFA1A3. The region has additional complexity in that each repeat unit contains a gene locus that can be occupied by one of two different genes, DEFA1 or DEFA3. These encode the human neutrophil peptides (HNPs) 1-3, antimicrobial peptides involved in the innate immune response. In order to understand the mutational processes and evolutionary history of a complex locus like DEFA1A3, spatial information is essential. Whilst haplotype DEFA1A3 copy numbers and haplotype ratios of DEFA1 vs. DEFA3 have been determined, little is known about the features shared by, and the structures of, related haplotypes. In this study, flanking sequence variation has been used to identify five classes of DEFA1A3 haplotype, which are tagged by four SNPs. Haplotypes within each class share similar features, such as DEFA1A3 copy number, but the associations differ between-class and between-population. Emulsion haplotype fusion-PCR has been used to determine the spatial arrangement of the DEFA1 and DEFA3 genes, as well as additional internal variants, across haplotypes of European ancestry. A comparison of the structures of related haplotypes suggests that the predominant mechanism of copy number change at the DEFA1A3 locus is intra-allelic rearrangements (i.e. between haplotypes from the same class), facilitated by the high sequence similarity of repeat units within each class. This explains the preservation of linkage disequilibrium across the DEFA1A3 locus. The relationship between DEFA1A3 copy number and gene expression is unclear. A comparison between DEFA1A3 haplotype class and HNP1-3 expression in a UK cohort suggests that DEFA1A3 haplotype structure does not influence gene expression. However, the identification of four SNPs which tag DEFA1A3 haplotype class and, in turn, haplotype structure in haplotypes of European ancestry, will aid further studies in this area.

The P2Y14 receptor is the most recently identified member of the P2Y family of receptors for adenine and uridine nucleotides and nucleotide sugars. It is activated by UDP, UDP-glucose and its analogues, as well as the synthetic analogue MRS2690, which exhibits greater potency and selectivity at the P2Y14 receptor. The principle aim of this study was to investigate the functional expression of the P2Y14 receptor in porcine pancreatic arteries, and the signalling pathways underlying the vasoconstriction evoked by P2Y14 receptor agonists, together with an examination of the effects of UDP-glucose and MRS2690 on insulin secretion from the rat INS-1 823/13 β-cell line. Segments of porcine pancreatic arteries were prepared for isometric tension recordings in warmed oxygenated Krebs’-Henseleit buffer. Agonists were applied after preconstriction with U46619, a thromboxane A2 mimetic. ATP induced vasoconstriction followed by a vasorelaxation in pancreatic arteries; the contraction was blocked by NF449 (a P2X1 receptor selective antagonist), while the relaxation to ATP was blocked by an adenosine receptor antagonist. Neither the contraction, nor the relaxation to ATP were affected by removal of the endothelium. ADP evoked vasorelaxation, which was inhibited in the presence of SCH58261 (a selective adenosine A2A receptor antagonist). UTP-induced vasoconstriction was attenuated significantly in endothelium-denuded arteries. UDP, UDP-glucose and MRS2690 induced concentration-dependent contractions in porcine pancreatic arteries with a rank order of potency of MRS2690 (10-fold) > UDP-glucose = UDP. The contractions evoked by UDP-glucose and MRS2690 were significantly attenuated in the presence of PPTN (a selective P2Y14 receptor antagonist), indicating actions at P2Y14 receptors. The expression of P2Y14-like receptor was shown by immunohistochemical and contractile studies to be on the endothelium of the pancreatic arteries. UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP production. UDP-glucose and MRS2690 increased the level of MLC2 phosphorylation; this effect was blocked by PPTN, indicating the involvement of P2Y14 receptors. UDP-glucose increased the level of ERK1/2 phosphorylation. UDP-glucose and MRS2690 inhibited glucose-induced insulin release from the rat INS-1 823/13 β-cell line; this effect was blocked by PPTN, indicating actions through P2Y14 receptors. PPTN itself was able to elevate significantly basal insulin secretion from INS-1 823/13 β-cells, which may suggest a constitutive release of UDP-glucose from these cells. These results suggest that, in porcine pancreatic arteries, ATP induces a vasoconstriction mediated by P2X1 receptors followed by a vasorelaxation evoked by adenosine receptors present on the vascular smooth muscle. ADP induced a relaxation mediated by adenosine A2A receptor. Moreover, my data indicate for the first time, an endothelium-dependent contraction evoked by UTP. A novel vasocontractile role of P2Y14 receptors in porcine pancreatic arteries was also documented. The contractile response was mediated largely by the endothelium. P2Y14-mediated contraction involves a cAMP-dependent mechanism, which is consistent with P2Y14 receptor coupling to Gi protein, and an elevation in phosphorylated MLC2 and ERK1/2. Activation of the P2Y14 receptor evoked a decrease in the level of insulin secreted from the rat pancreas. The current data have identified novel roles of the P2Y14 receptor as a mediator of pancreatic artery contractility and in regulation of insulin secretion. If its role within the vasculature is shown to be more widespread, the P2Y14 receptor may be a novel target for the treatment of cardiovascular disease.

The potential application for stem cell therapy is vast. Despite a limited understanding of their mechanisms of action, clinical trials assessing stem cells in human stroke are underway. Colony stimulating factors (CSF) such as granulocyte colony stimulating factor (G-CSF), which have been used to mobilise haematopoietic stem cells (HSC), also show promise in treating stroke. Preclinical experiments evaluating the effect of G-CSF in stroke were meta-analysed; G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Further studies assessing dose-response, administration time, length of ischaemia and long-term functional recovery are needed. Tracking iron-labelled cells with MRI may help to establish migratory patterns following transplantation. Our systematic review of iron-labelled stem cells in experimental stroke suggests that compounds already licensed for humans (ferumoxide and protamine) may potentially be used in clinical trials. In a phase IIb single-centre randomised controlled trial (n=60), the safety of G-CSF in recent stroke was assessed (STEMS-2). G-CSF appears safe when administered subacutely and may reduce stroke lesion volume. Phase III trials are required to test efficacy. An updated Cochrane review on CSFs in stroke shows that G-CSF was associated with a non-significant reduction in early impairment but had no effect on functional outcome in 6 small studies. In two trials, erythropoietin therapy was significantly associated with death by the end of the trial. It is too early to know whether G-CSF could improve functional outcome in stroke. In 8 recruits randomised into STEMS-2, mobilised CD34+ HSCs were paramagnetically labelled, re-infused and tracked with serial T2* MRI. Post-stroke HSC labelling appears safe and feasible. There is suggestive evidence in one patient that labelled HSCs migrate to the ischaemic lesion. Our in vitro evaluation of CD34+ HSCs has revealed that uptake of superparamagnetic iron oxide (SPIO) is enhanced but not dependent upon a transfection agent. Iron labelling of CD34+ cells in this manner did not affect cell viability or inhibit growth. This methodology could be applied to clinical trials. We have established the expression of G-CSF protein, its receptor (G-CSFR) and CD34 antigen in post-mortem brain tissue from participants recruited to STEMS-2. Areas of angiogenesis and expression of G-CSFR in acute and chronic infarction suggest potential targets for therapy. There are many preclinical studies reporting the effects of stem cells in treating stroke (with a noticeable lack of neutral or negative articles). Despite the wealth of literature there remain many unanswered questions and patients should not undergo stem cell therapy unless it is as part of a well designed clinical trial.

Satellite cells are muscle-specific progenitor cells involved in the routine maintenance of skeletal muscle homeostasis, growth and regeneration. They are activated by various stimuli (myotrauma, growth factors etc), undergo rounds of proliferation as skeletal muscle myoblasts, to differentiate and fuse with each other to generate new myotubes or onto existing myofibres to augment growth or repair damaged fibres. Satellite cells contribute to hypertrophy by facilitating nuclear addition, which maintains contractile protein synthetic capacity. Conversely, during atrophy the dysregulation of satellite cells (e.g., via myogenic suppression), causes an opposing deficit in nuclear supplementation/contractile protein synthesis. The ‘activity status’ of satellite cells, an important determinant of muscle regenerative capacity is not routinely addressed in studies characterising mechanisms of muscle hypertrophy and atrophy. Therefore, the investigations described within this thesis examined the satellite cell specific signalling events that contribute to muscle loss or gain, in rodent models experiencing non-mechanically-induced muscle hypertrophy or atrophy. Chronic administration of an anabolic agent (BRL-47672, the pro-drug of clenbuterol) increased the expression of early components of satellite cell myogenesis (pax7, ki-67, myoD) but caused no alteration in myogenin expression, relative to control in rat soleus muscle. Pro-drug administration increased myostatin expression, with no concomitant change in follistation mRNA; this is likely a compensatory mechanism to check excessive muscle growth. These results provided evidence of increase satellite cell activity in hypertrophying muscle. In a lipopolysaccharide (LPS)-infusion model of muscle atrophy, satellite cells were inhibited in an inflammatory-dependent manner. LPS infusion caused early (<2hr) elevations inflammatory cytokines TNF-, IL-6 and NF-B. LPS-induced elevation in cytokine transcript levels paralleled increased myostatin and decreased pax7 and myoD mRNA and protein expression. The differential increase in cytokines also paralleled the reduction in the number of pax7+ and myoD+ satellite cells. These results suggest that alterations in satellite cell activity may contribute to the progression of muscle atrophy, due to the suppression of muscle compensatory mechanisms, which include satellite cell activation, differentiation and fusion for nuclear supplementation. Co-infusion with an anti-inflammatory agent, dexamethasone (Dex), blunted LPS-induced increase in inflammatory cytokines but had an additive effect on myogenic suppression. Dex+LPS infusion prevented LPS-induced increase in myogenin and resulted in an additional suppression of pax7 and myoD, greater than that elicited by either substance alone. Negative regulation of satellite cells by glucocorticoids could impede their efficacy in the treatment of inflammatory muscle disorders. The research within this thesis emphasise satellites are important for maintenance of muscle homeostasis and their activation/inhibition, may determine the magnitude of muscle loss or gain. This was demonstrated by the pattern of pax7 and myoD expression in hypertrophying muscle, where both markers were up-regulated and in atrophying muscle, where they were down-regulated. Down-regulation of these markers in atrophy could have implications for muscle regenerative capacity, especially myoD, whose expression was continuously inhibited across all time-points sampled in septic muscles. Satellite cells are a major source of compensatory action in skeletal muscle, their activation and subsequent myogenesis represents an auxiliary mechanism by which muscle responds to damaging stimuli; therefore their dysregulation (through the alteration of key myogenic markers) results in an alteration of normal function. Such dysregulation, as frequently reported in cases or progressive muscle degeneration and sarcopenia, limits the efficacy of muscle compensatory processes (i.e. satellite cell activation/proliferative or differentiation potential), thereby contributing to the progression of muscle atrophy and myopathy.

Research on human genetic variation has shown that the human genome is not a fixed, invariant framework, but that there can be extensive structural variation. This variation includes copy number variation (CNV), which can lead to changes in DNA dosage contributing significantly to variation between individual human genomes and heritable traits. Human beta-defensins are small, secreted antimicrobial peptides encoded by DEFB genes located in a cluster of at least seven genes on 8p23.1. These genes are highly variable in copy number but accurate measurement of multiallelic copy number variants is challenging, particularly for high copy numbers, and has not been intensively studied until recently. A new PRT-based (Paralogue Ratio Test) triplex assay was developed to accurately measure the multiallelic beta-defensin copy number variation. The Triplex assay was demonstrated to be an accurate and powerful method to measure copy number variation in large case-control association studies. This method was used to study the beta-defensin CNV in psoriasis disease, showing that high beta-defensin copy number is associated with susceptibility to psoriasis in Caucasians. Studying population variation of CNV showed that variation in copy number of beta-defensin is not significantly different across human populations. To understand the evolutionary history of beta-defensin CNV in the primate lineage, the study of CNV at this locus was carried out in great apes. Beta-densin genes are copy variable in human and chimpanzee, but not in gorilla, suggesting that variation in copy number of beta-defensin genes may have arisen in the human-chimpanzee lineage after the divergence with gorilla.

The rat was used as a model for the assessment of a high fat diet (HFD) and HFD/streptozotocin (STZ) induced Type II diabetes upon lipid deposition and development of inflammation in metabolically active tissues. HFD feeding for a period of 10 weeks did not induce significant weight gain in animals compared to those fed on normal chow (NC). There was also no significant effect of HFD feeding upon blood glucose and insulin levels. Adipose and skeletal muscle tissues showed minimal effects of HFD feeding at both the histological and molecular level. Histological assessment of liver tissue revealed marked steatosis in HFD fed animals. Molecular studies showed that genes involved in lipid and glucose metabolism and insulin signalling were decreased while genes involved in endoplasmic reticulum (ER) stress were elevated. Liver triglyceride fatty acid profiles resembled those of the diet with no significant differences in lipoprotein triglyceride levels observed between experimental groups. STZ injection induced hypoinsulinemia and hyperglycaemia. The changes observed at the molecular level were related to insulin depletion. Pioglitazone intervention did not cause any major changes in the STZ treated animals. The main conclusion was that HFD induces liver steatosis due to increase lipid flux from the diet despite the absence of weight gain or increased adipose tissue or skeletal muscle lipid content. This suggests that consumption of a high fat diet may cause the development of fatty Liver disease in the absence of weight gain or overt obesity.

N-methyl-D-aspartate receptors (NMDAR) are ionotropic glutamate receptors which can be blocked by Mg2+ in a voltage-dependent manner and are highly permeable to Ca2+, hence they represent a medically relevant target for neurodegenerative disorders caused by excitotoxicity. The two main objectives of this study were, (i) to determine the impact of Q/R/N, +1 and -8 sites modification in the M2 pore region of GluN2A NMDAR subunit on Mg2+ block and other open channel blockers; and (ii) to evaluate novel multi-target-directed ligands (MTDL) for Alzheimer’s disease therapy. The Xenopus laevis oocyte expression system was employed where NMDAR subunit cRNAs were injected into the oocytes and responses to NMDA/glycine and channel blockers were recorded using two-electrode voltage clamp (TEVC) electrophysiology. Pore region mutations to investigate the impact of Q/R/N and adjacent sites were characterized using Mg2+, memantine, MK-801, philanthotoxin analogues and an MTDL compound, CR18. NN at the Q/R/N and +1 sites in GluN2A subunits were mutated to GR and RR, while W at the -8 position (in relation to the Q/R/N site), was mutated to N. Wild type and mutated GluN2A were co-expressed with GluN1-1a in Xenopus oocytes and antagonistic responses by channel blockers were recorded with TEVC. At -75 mV, the RR mutation significantly increased IC50s of Mg2+, memantine and MK-801 by 27-, 42- and 325-fold respectively, compared to wild-type. As for the GR mutation, IC50s were also significantly increased for memantine and MK-801 by 5- and 132-fold respectively, compared to wild type. W to N mutation at the -8 position did not significantly affect blocking potencies for all channel blockers. Blocking potency for PhTX-343 was not significantly altered by any mutations. This study provided evidence that the presence of G and R at the Q/R/N and +1 sites are likely responsible for the changes in blocking sensitivity and play important roles in ion permeability. The fact that PhTX-343 remained potent despite the mutations suggest that this compound might have a different mode of action or different binding site other than the M2 region and should be further characterized. In the MTDL study, twenty one novel compounds were tested on GluN1-1a/GluN2A NMDAR subunits. Thirteen were memantine-derivatives (MAB) incorporated with antioxidant moieties, three were spermine-derived polyamines also incorporated with antioxidants, and five were combinatorial forms of donepezil and carvedilol. The antagonistic properties of the compounds were tested electrophysiologically at -60 mV and compared with Mg2+ and memantine. The MAB series were found to be weak NMDAR channel blockers suggesting the loss of memantine functionality due to attachment of the antioxidant structure to its amine group. Subsequently, modification of the linker point to memantine moieties to free its amine group eventually resulted in weaker NMDAR channel blockers with IC50s of more than 100 µM. The spermine-derived polyamines (CR compounds) were potent NMDAR blockers with IC50s (0.69 to 2.35 µM) comparable to memantine (2.28 µM) and significantly lower than Mg2+ (10.1 µM) and also exhibited voltage-dependence block. Our mutation study revealed that CR18, the most potent MTDL compound was less sensitive in NMDAR containing GR or RR mutation in GluN2A subunits. This is a favourable property of an NMDAR blocker for potential Alzheimer’s disease treatment since GluN3 subunits containing GR or RR at the Q/R/N and +1 sites are less permeable to Ca2+ influx and has been shown to exert neuroprotective effects.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen belonging to the Enterobacteriaceae and considered to be a leading cause of acute infantile diarrhoea in developing nations. During onset of infection, EPEC inject a variety of effector proteins into the host epithelial cells using the type III secretion system (T3SS). These effectors are encoded within the pathogenicity island (PAI) called the locus of enterocyte effacement (LEE). EPEC also secretes the non-LEE encoded serine protease EspC (EPEC secreted protein C) into the extracellular milieu. This autotransporter (AT) exits EPEC through a type V secretion system (T5SS) and is subsequently delivered into host cells by the T3SS. The precise steps by which EspC is secreted are still unknown. By using both traditional and advanced optical microscopes, it is shown here that EspC tagged with two different fluorescent labels locates to a structure that resembles the spiral bacterial cytoskeleton when it has its β-barrel domain attached. It was discounted that the spiral formation was an artefact of aggregation. Moreover, production of another AT (AaaA, derived from Pseudomonas aeruginosa) also generated spiral structures that resemble the ones observed with EspC. Perturbing the structure of the bacterial cytoskeleton actin homologue MreB with the S-(3,4-dichlorobenzyl) isothiourea compound (A22) disrupted the localization of EspC-mCherry. Furthermore, producing EspC-mCherry in an E. coli SecA mutant generated altered localization patterns. Collectively, these results indicate that the spiral localization of EspC is dependent upon its C-terminal β-barrel domain, the Sec translocon and the actin homologue MreB and this spiral secretion pathway seems to be conserved among ATs. Further analysis is required to reveal the molecular mechanism underlying spiral formation to unravel the mystery of AT secretion, thereby enabling the development of new therapeutic targets and treatments.